Sequence-specific inhibition of small RNA function

Abstract

The present invention relates to the discovery of a method for inhibiting RNA silencing in a target sequence-specific manner. RNA silencing requires a set of conserved cellular factors to suppress expression of gene-encoded polypeptide. The invention provides compositions for sequence-specific inactivation of the RISC component of the RNA silencing pathway, and methods of use thereof. The RISC inactivators of the present invention enable a variety of methods for identifying and characterizing miRNAs and siRNAs, RISC-associated factors, and agents capable of modulating RNA silencing. Therapeutic methods and compositions incorporating RISC inactivators and therapeutic agents identified through use of RISC inactivators are also featured.

What is claimed is:  
     
       1. A method for inhibiting RNA silencing of a gene, comprising, contacting a mammalian cell or organism containing an exogenous siRNA duplex that directs RNA silencing of the gene by RISC-mediated cleavage, with a single-stranded, nuclease resistant RNA oligonucleotide RISC inactivator between about 10-40 nucleotides in length, wherein the RISC inactivator comprises a nucleotide sequence sufficiently complementary to a guide strand sequence of the siRNA duplex to inhibit RNA silencing of the gene by the siRNA duplex, wherein the RISC inactivator comprises modified ribonucleotides, and wherein the RISC inactivator is a stochiometric, irreversible inhibitor of RISC function, such that RNA silencing of the gene is inhibited, wherein the RNA silencing is RNA interference. 
     
     
       2. The method of  claim 1 , wherein the RISC inactivator comprises between about 10-40 modified ribonucleotides. 
     
     
       3. The method of  claim 1 , wherein the RISC inactivator comprises between about 15-20 modified ribonucleotides. 
     
     
       4. The method of  claim 1 , wherein the RISC inactivator comprises between about 20-25 modified ribonucleotides. 
     
     
       5. The method of  claim 1 , wherein the RISC inactivator comprises between about 25-30 modified ribonucleotides. 
     
     
       6. The method of  claim 1 , wherein the RISC inactivator comprises between about 30-35 modified ribonucleotides. 
     
     
       7. The method of  claim 1 , wherein the RISC inactivator comprises between about 35-40 modified ribonucleotides. 
     
     
       8. The method of  claim 1 , wherein the RISC inactivator is administered at about 0.1-20nM doses. 
     
     
       9. The method of  claim 1 , wherein the RISC inactivator is administered at a dose of less than about 500 nM. 
     
     
       10. The method of any one of  claims 1 ,  2  and  3 - 7 , wherein the RISC inactivator 2′-O-methyl modified nucleotides. 
     
     
       11. The method of any one of  claims 1 ,  2  and  3 - 7 , wherein the RISC inactivator comprises locked nucleic acid (LNA) modifications. 
     
     
       12. The method of any one of  claims 1 ,  2  and  3 - 7 , wherein the RISC inactivator comprises phosphorothioate modifications. 
     
     
       13. The method of any one of  claims 1 ,  2  and  3 - 9 , wherein the RISC inactivator comprises a nucleotide sequence which is perfectly complementary to the guide strand of the siRNA duplex.